Project Summary/Abstract Overview of Research in the Lorigan Lab and 5 Year Goals (Overview): Currently, we have limited structural information on membrane proteins. The Lorigan lab is interested in developing new biophysical methods to probe the structural and dynamic properties of integral membrane proteins using state-of-the-art pulsed EPR spectroscopic techniques and membrane solubilizing polymers. The overall objective is to study membrane proteins with EPR in a lipid bilayer as opposed to a micelle or detergent because it more closely mimics a cell membrane. Several proteins have been shown to not function or fold up correctly in a micelle when compared to a lipid bilayer. This is challenging because it is more difficult to express, purify, and conduct biophysical spectroscopic experiments on membrane proteins when compared to micelle or globular systems. My expertise in membrane protein EPR and sample preparation coupled with the powerful pulsed EPR instrumentation (DEER and ESEEM) in my lab that can measure long range distances has attracted several significant collaborators with important biological problems. My research lab works directly with several researchers to dramatically improve the quality of membrane protein sample preparation to yield high quality DEER data that leads to more accurate structural information. Please see the letters of support. The major biological focus of the lab is on membrane protein channels that are directly related to heart disease. KCNQ1 (Q1) is a biologically significant voltage gated potassium channel found in the heart that is modulated by the membrane protein KCNE1 (E1). KCNQ1/KCNE1 interactions slow down the activation kinetics of KCNQ1 required for proper channel and heart function. Hereditary mutations in Q1/E1 can cause Long-QT syndrome, atrial fibrillation, sudden infant death syndrome, cardiac arrhythmias, and congenital deafness. Q1 is a membrane protein with six transmembrane (TMD) helices, the first four TMDs form the voltage sensor domain Q1-VSD (S1-S4), linked to the pore domain (S5-S6) by the S4-S5 linker and the cytosolic N and C-terminal domains. The three- dimensional structure of KCNQ1 or the E1/Q1 complex has not been determined. Furthermore, the structural nature of the binding interaction/mechanism of E1 with Q1 is poorly understood and has only been investigated indirectly with biochemical binding and cross-linking assays. We are currently applying state-of-the-art EPR techniques to directly probe the structural and dynamic properties of Q1 and the E1/Q1 complex. The following pertinent biological questions will be answered: Which segments of KCNQ1 are helical in a lipid bilayer? What is the structure and topology of the KCNQ1 with respect to the membrane? How does Q1 bind and interact with the E1 protein that is required for function? ! (5 Year Goals of the Lab): (1) Develop new biophysical techniques to study the structure and dynamics of membrane proteins; (2) Investigate the structure and topology of the KCNQ1 K+ channel; (3) Elucidate the binding mechanism of KCNQ1 with KCNE1; and (4) Apply the membrane protein techniques that we develop to investigate the structure of several biologically important integral membrane proteins (influenza tetrameric M2 protein, Pentameric ligand-gated ion channels (pLGIC, TRPM8 and PIRT) using pulsed EPR spectroscopy (DEER and ESEEM). Transformative biophysical techniques will be developed to study the structural and dynamic properties of membrane proteins. These state-of-the-art pulsed EPR spectroscopic techniques will move the field forward by dramatically increasing sensitivity, accuracy of distance measurements for all membrane protein systems. Also, a new polymer based membrane mimetic system will be developed that will enable researchers to more easily conduct structural and functional measurements of membrane proteins in a lipid bilayer. The size of the lipodisqs can be fine-tuned by the polymer to match the size of the membrane protein complex.